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DNA as a Nanostructure

The central dogma:

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The components of DNA and RNA:

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5’-ACG -3’

DNA

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DNA is double stranded -> double helix (2º structure)

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The Minor and Major Groove of DNA

Because the 2 glycosidic bonds are not diametrically opposite each other-> each base pair has a larger side -> the major groove -> and a smaller side -> the minor groove

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Dimensions of DNA

Adjacent bases are separated by 3.4 Å

Helix repeats every 34 Å

10 bases per turn of helix

Diameter of helix is 20 Å

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Z- form

Different forms of DNA

Short oligonucleotides that have alternating pyrimidines and purines -> CGCGCGC

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Other DNA structures than double helix - Quadruplex

Crystal structure of parallel quadruplexes from human telomeric DNA. The DNA strand (blue) circles the bases that stack together in the center around three co-ordinated metal ions (green). (By Thomas Splettstoesser)

Just with Purin rich strands (G)

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Other DNA structures than double helix - Quadruplex

The arrangement of guanine bases in the G-quartet, shown together with a centrally placed metal ion. Hydrogen bonds are shown as dotted lines, and the positions of the grooves are indicated. (b) The poly(dG) four-fold, right-handed helix. (c) Surface view representation of a quadruplex structure comprising eight G-quartets, with the central channel exposed to show an array of metal ions (coloured yellow).

-> Microelectronics

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Relaxed and Supercoiled DNA (3º structure)

Supercoiled DNA -> Relaxed DNA

-> 1 DNA strand needs to be nicked

1.Enzymes (Topoisomerases)2.Sheer forces or chemicals

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-> The twist is the number of helical turns in the DNA

-> the writhe is the number of times the double helix crosses over on itself (these are the supercoils).

The relationship of twist, writhe and supercoiling (Linking number)is expressed as the equation:

S = T + W

Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling.

DNA Supercoiling

-> Overtwisting leads to   postive supercoiling-> undertwisting leads to negative supercoiling

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Linking Number Determines the Degree of

Supercoiling

linking number -2

linking number -1 linking number 0

linking number 1 linking number 2 linking number 3

Linking Number describes the linking of two closed curves in three-dimensional space

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Linking Number Determines the Degree of

Supercoiling

Most DNA molecules are neg. supercoiled

Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling.

-> Overtwisting leads to   postive supercoiling-> undertwisting leads to negative supercoiling

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Supercoiling in eukaryotic linear DNA (Chromosomes)

-> Supercoiling happens between Histone proteins

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Nucleic Acid Synthesis - Polymerization

DNA polymerase -> Replication:

DNA -> DNA

5’ -> 3’

Primer

Proof reading

RNA polymerase -> Transcription:

DNA -> RNA

5’ -> 3’

No primer

No proof reading

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DNA Replication,

Recombination, and Repair

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DNA replication

Replication is: -> bidirectional

-> semiconservative

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DNA Replication

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Topoisomerase I structure/mechanism

Topoisomerases prepare double helix for unwinding -> change “linking number”

1. Preparation for unwinding requires neg. supercoiled DNA (TopoII)

2. In the process of unwinding -> DNA needs to be relaxed (TopoI)

-> Important for Replication, Transcription, Recombinantion

TopoI -> cleaves one strand -> relaxes neg. supercoiled DNA

No Energy (ATP) required !!

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Topoisomerase II structure/mechanism (DNA gyrase)

TopoII -> cleaves both strands -> introduces negative supercoiling

-> Requires Energy (ATP) !!!

Gyrase Inhibitors

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E. coli DNA polymerase

PolymeraseActivityα-subunit

ε-subunit

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DNA polymerase mechanism

One Mg2+ coordinates the 3’-OH group of the primer -> OH-group of primer attacks P-group of nucleotide

Polymerase donates 2 H-bond to base pair in minor groove

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Shape selectivity

Binding of NTP induces conformational change -> generating a tight pocket for base pair

-> Conformational change just when incoming dNTP fits to template DNA

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Proofreading

ε Subunit of E. coli Polymerase III (responsible for sythesis of DNA) has 3’-> 5’ exonulease activity

The growing chain moves sometimes to the exonuclease site -> checking if incorrect nucleotide is incorporated -> wrong nucloetide is hydrolysed

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Helicase structure/mechanism

Used for unwinding of DNA -> replication

It has 4 domains:A1 domain has a P-loop NTPase -> bind ATP

B1 + A1 bind ss DNABinding of ATP -> conformational change of P-loop -> closure of cleft between B1 + A1 -> A1 releases DNA -> A1 slides along DNA -> moving closer to B1

Release of ADP -> cleft between B1 + A1 opens-> A1 still bond tighter to DNA -> DNA pulled across B1 towards A1

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Origin of Replication (E. coli)

Binding and assembly of DnaA initiates replication

Surrounding of Ori -> AT rich -> important for local melting of DNA

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Prokaryotes

Eukaryotes

Initiation of replication: Origins

In Prokaryotes: 1 folk/circular chromosom -> 1000 bp/sec -> 42 min to replicate E. coli chromosome (4.6 mill bp)

In Eukaryotes: In humans -> 100 bp/sec per folkSize: 3x109 bp in 23 chromosomes

Assume the same as in prokaryotes: 1 folk per chromosome -> 23 folks -> 1.3x108 bp/folk with a speed of 100 bp/sec 1 Replication cycle would take 1.3x106 sec -> 362 h-> 15 days !!!!

-> in real: 1 replication cycle is 8 h-> 30.000 folks (not all always active)

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Elongation of Replication

Introduces neg. supercoiling

DNA polymerase I degrades RNA primerDNA ligase closes the fragments

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DNA polymerase complex -> Replisome

DNA polymerase III core enzyme -> dimer

DNA enclosing site

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Coordination Between the Leading and the Lacking Strand

-> Replication is bidirectional

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Telomeres

In the process -> quadruplex structures are formed

Telomer end are involved in aging of cell !!

Eukaryotes need Telomer ends -> otherwise chromosomes would shrink each replication cycle

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Recombination

Essential in the following processes:

-> When replication stops -> recombination reset replication process

-> When DNA strands break -> recombination repairs DNA

-> In meiosis -> genetic diversity (cross over events)

-> generation of diversity for antibodies (Exon shuffling)

-> viruses use recombination to integrate into genome

-> in recombinant technologies -> generate recombinant organisms (knock-out mice)

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RecA protein initiates recombination by strand invasion

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Recombination mechanism – Holliday Junction

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Transition: AT <-> GC

TA <-> CG

Exchange of Purine by Purine and Pyrimidine by Pyrimidine

Transversion: AT <-> TA

AT <-> CG

TA <-> GC

CG <-> GC

Exchange of Purine by Pyrimidine and the other way round

Mutations – changes in DNA sequence

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Tautomerization: The conversion of two isomers that differ only in the position of protons (and often double bonds)

Mutation by Tautomerization

-> Transition from AT -> GC

Analog to thymine

-> Transition from TA -> CG

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Chemical Mutagens

Conversion of A -> Hypoxanthine

Hypoxanthine pairs with C

Transition from AT -> GC

Acridines: Induce frame shift by intercalating into DNA leading to incorporation of additional bases

(Ethidium bromide)

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Chemical Mutagens

Produced by fungi;

Activated by Cyt P450

Modifies bases such as G

Active epoxide

-> Transversion from GC -> TA

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Deamination of modified Cytosine

5-methylcytosine -> hot spot for mutations

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-> Thymine dimer

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DNA repair pathways

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Excision repair of thymine dimers

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Uracil repair

U in DNA

formed by deamination of cytosine

-> excised and replaced by C

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Mismatch repair